This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed an approach for identifying and quantifying oxidant-sensitive protein thiols using a cysteine-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, Foster City, CA). We are now using this approach to explore the relationship between redox sensitivity of individual cysteine residues and physiologically significant oxidative post-translational modifications, as well as irreversible thiol oxidation by oxidant stress associated with disease. As a proof of principle, we have quantitatively evaluated oxidative post-translational modifications of the recombinant protein H-Ras that accompany changes in its activity. H-Ras (1-10 ug) was treated with peroxynitrite (ONOO-, 100 uM) for 5 min. at 37 deg C in the presence or absence of reduced glutathione. Ras activity was assayed by association with Raf-1 and by GTP/GDP exchange. The ONOO--treated samples were labeled with heavy ICAT reagent by incubating at 37 deg C for 30 min. The heavy isotope-labeled protein was mixed in equal amounts with untreated H-Ras that had previously been labeled with light ICAT reagent. The light and heavy labeled proteins were digested with trypsin, desalted, and affinity-purified using an avidin cartridge (Applied Biosystems, USA). The samples were concentrated and analyzed by MALDI-TOF MS and capLC-MS/MS. The amino acid sequence of H-Ras contains six cysteine residues. Of the six, four (118, 181, 184 and 186) are surface-exposed, as determined by structural, chemical and mutational studies. Although Cys-181, Cys-184 and Cys-186 are known to be modified by prenylation in intact cells, all of the reactive cysteines are potentially oxidized during normal and pathological conditions and this oxidation could alter the cellular function of the protein. We recently demonstrated that Ras was S-glutathiolated and activated by oxidants generated from NADPH oxidase in smooth muscle cells stimulated with angiotensin II. This result now makes it imperative to quantify the thiol modifications in the protein associated with its oxidant-mediated activation. The activity of Ras was significantly increased 2- to 3-fold following exposure to peroxynitrite and glutathione, but not to peroxynitrite alone. We therefore applied our ICAT approach to identify and quantify cysteine modifications that occur upon treatment with ONOO- in the presence and absence of glutathione. MALDI-TOF MS of the ICAT-labeled peptides of H-Ras showed 15-20 ICAT-labeled peptides with the appropriate 9-Da difference between the light and heavy ICAT-labeled peptides. LC-MS was used to quantify the degree of cysteine oxidation on the basis of the change in signal intensity for the heavy ICAT-labeled peptide. In the ONOO--treated samples, the Cys-118 is oxidized 47%, as measured from the change in the intensity for the heavy-labeled peptide, whereas the non-reactive Cys 80 is not oxidized as indicated by no change in the intensity for the heavy ICAT-labeled peptide. We anticipate that quantitative evaluation of the extent of modification of individual cysteine residues can be correlated to the activation or inactivation of H-Ras when subjected to reactive oxygen/nitrogen species. We have thus successfully applied our ICAT approach to quantitatively evaluate the oxidative PTMS of the protein H-Ras that accompany changes in its activity. We are extending this approach to include other proteins that are known (or thought) to play important roles in oxidative stress related to cardiovascular disease. including Sirt1. A method for efficient expression of Sirt1 has been developed and the protein and selectively modified forms are being generated so that structure/activity relationships can be determined.